Method of inhibiting ethylene production in plants

ABSTRACT

Methods of inhibiting ethylene production in a plant are provided that involve contacting a plant or plant part with an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). The methods can be used to delay fruit and vegetable ripening, flower opening, or senescence of cut flowers. Also described are kits including an ACCO inhibitor and a floral preservative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application of InternationalApplication No. PCT/US2006/008584, filed Mar. 10, 2006, which claimspriority to U.S. Provisional Patent Application No. 60/660,809, filedMar. 11, 2005, the contents of which are hereby incorporated byreference into the present disclosure in their entirety.

GOVERNMENT SUPPORT

This subject matter described herein was made with government supportunder GM 25765 awarded by the National Institutes of Health. Thegovernment has certain rights in the subject matter described herein.

FIELD

Described herein are methods for controlling the rate of ripening andsenescence of economically valuable plants and plant parts.Specifically, described herein are methods of inhibiting ethyleneproduction in plants and plant parts, and particularly to inhibiting theenzymatic activity of 1-aminocyclopropane-1-carboxylate oxidase (ACCO)in cut flowers, fruits, and vegetables.

BACKGROUND

A major problem that faces the agriculture industry is timing theripening of fruits, vegetables and the opening of cut flowers so thatsuch products are not spoiled prior to sale or use. Spoilage accountsfor significant losses in agricultural and floriculture markets.

Cut flowers represent a substantial commercial market in the U.S.; thewholesale market is over $400 million. Both growers and retailers offlowers suffer significant losses due to flower spoilage and limitedshelf life. Growers typically cut flowers prior to flower opening andstore them in a cool environment to delay opening. The flower stems alsoare typically placed in a preservative solution. Such preservativesgenerally have a bleach-based biocide, an acid component and a sugar.However, even with such approaches, cut flowers generally can be storedfor only short periods of time.

Additionally, there is a tremendous need to improve the shelf life ofharvested fruits and vegetables. A significant portion of spoilage ofharvested produce is due to accelerated production of ethylene by theharvested plant part. Currently, water spraying, cold storage andsequestering from other ethylene-producing produce items are methodsused to control ripening of fruits and vegetables after harvest. Anothercurrent method is treatment with ethylene analogs which bind to theethylene receptor.

Ethylene is an important signaling molecule in plants and influences avariety of processes including plant growth, fruit ripening, and flowerdevelopment and senescence. Ethylene is synthesized in plants through aseries of enzymatic transformations. S-adenosylmethionine (SAM) isconverted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACCsynthase. Adams, et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:170-174.ACC oxidase then oxidizes ACC to form ethylene. Yang, et al. (1984)Annu. Rev. Plant. Physiol. 35:155-189.

Previous approaches to ethylene biosynthesis in plants have included useof an antisense construct that inhibits expression of a component of theethylene biosynthesis pathway. Hamilton, et al. (1990) Nature346:284-287 report the use of an antisense TOM13 (ACC oxidase) gene intransgenic plants. Picton et al. (1993) Plant Journal 3:469-481, reportaltered fruit ripening and leaf senescence in tomatoes expressing anantisense against the ethylene-forming enzyme. Yet another approach isto transgenically introduce an enzyme that degrades ACC prior toethylene formation (U.S. Pat. No. 6,271,009). Chemical inhibitors alsohave been used including those disclosed in U.S. Pat. Nos. 4,957,757 and4,851,035. Lunkenheimer and Lürssen describe1-methylamino-cyclopropane-1-carboxylic acid derivatives as plant growthregulators (U.S. Pat. No. 5,041,612). Bregoli, et al., report the use ofaminoethoxyvinylglycine (AVG) as an inhibitor of ACC synthase (the priorenzyme in the ethylene pathway in plants) but its utility is limited bythe high cost of product and its limited effectiveness. PhysiologiaPlantarum, Vol. 114 Issue 3, Page 472—March 2002.

There still is a need for improved methods of preventing prematureripening of plants and plant parts while in transit to the market thatcan be applied to a wide variety of plants.

SUMMARY

Described herein are methods of inhibiting ethylene production in aplant or plant part. Methods described herein include contacting a plantor plant part with an effective amount of an ACC derivative having theformula:

-   -   where R₁, R₂, and R₇ may be as follows

(a) R₁ and R₇ are independently selected from H, —OH, substituted orunsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substituted or unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted or unsubstitutedC₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, or substitutedor unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; andR₂ is —OH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl, —OR³, —NR⁴R⁵, or —SR⁶,except R₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy,cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H or an acylgroup and R₇ is methyl or R₁ is methyl and R₇ is H or an acyl group;where R³ is —H, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R⁴, R⁵ and R⁶ areindependently selected from the group consisting of —H, substituted orunsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substituted or unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted or unsubstitutedC₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, or substitutedor unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl.

(b) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and R₂ is—OH, —NH₂, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; —OR³, —NR⁴R⁵, or —SR⁶, exceptR₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy,—NH₂, alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ ismethyl or R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₁-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; and R⁴, R⁵ and R⁶ areindependently selected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; or C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy.

(c) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; and R₂ is —OH, —NH₂, unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, and methoxy; C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; C₆-C₁₀ aryl substituted with a group selectedfrom halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenylsubstituted with a group selected from halo, hydroxyl, and methoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,and methoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenylsubstituted with a group selected from halo, hydroxyl, and methoxy; andR⁴, R⁵ and R⁶ are independently selected from —H, unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy.

(d) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆ cycloalkyl or C₃-C₆cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; and unsubstitutedC₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R₂ is —OH, —NH₂,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; or unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; and R⁴, R⁵ and R⁶ are independently selected from—H, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; and unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl.

(e) R₇ is —H; R₁ is selected from H, substituted or unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl,substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl,substituted or unsubstituted C₄-C₉ heteroaryl, or substituted orunsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R₂is —OH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl,substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl,substituted or unsubstituted C₆-C₁₀ aryl, substituted or unsubstitutedC₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted or unsubstituted C₄-C₉heteroaryl, or substituted or unsubstituted C₄-C₉ heterocycloalkyl orC₄-C₉ heterocycloalkenyl, —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substitutedor unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted orunsubstituted C₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkylor C₆-C₁₄ aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, orsubstituted or unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; and R⁴, R⁵ and R⁶ are independently selected fromthe group consisting of —H, substituted or unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl.

(f) R₇ is —H; R₁ is selected from H; unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and R₂ is—OH, —NH₂, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; —OR³, —NR⁴R⁵, or —SR⁶, exceptR₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy,—NH₂, alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ ismethyl or R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; and R⁴, R⁵ and R⁶ areindependently selected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; or C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy.

(g) R₇ is —H; R₁ is selected from H; unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; and R₂ is —OH, —NH₂, unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, and methoxy; C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; C₆-C₁₀ aryl substituted with a group selectedfrom halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenylsubstituted with a group selected from halo, hydroxyl, and methoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,and methoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenylsubstituted with a group selected from halo, hydroxyl, and methoxy; andR⁴, R⁵ and R⁶ are independently selected from —H, unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy.

(h) R₇ is —H; R₁ is selected from H; unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₆ cycloalkyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; and unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; and R₂ is —OH, —NH₂, unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl; unsubstituted C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; —OR³, —NR⁴R⁵, or —SR⁶,except R₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy,cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H or an acylgroup and R₇ is methyl or R₁ is methyl and R₇ is H or an acyl group;where R³ is —H, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl;unsubstituted C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl; unsubstitutedC₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl;unsubstituted C₄-C₉ heteroaryl; or unsubstituted C₄-C₉ heterocycloalkylor C₄-C₉ heterocycloalkenyl; and R⁴, R⁵ and R⁶ are independentlyselected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl;unsubstituted C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl; unsubstitutedC₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl;unsubstituted C₄-C₉ heteroaryl; and unsubstituted C₄-C₉ heterocycloalkylor C₄-C₉ heterocycloalkenyl.

(i) any of the ACC derivatives described above with the heteroarylselected from pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazoyl,pyridyl, pyrazinyl, pyrimidineyl, furyl, and thienyl.

(j) any of the ACC derivatives described above with the heteroarylselected from pyrrolyl, pyridyl, and thienyl.

(k) any of the ACC derivatives described above with the heteroarylpyrrolyl.

(l) any of the ACC derivatives described above with R₂ hydroxyl, amino,or lower alkoxy, except R₂ is not —OH, substituted alkoxy, alkenyloxy,alkinyloxy, cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H oran acyl group and R₇ is methyl or R₁ is methyl and R₇ is H or an acylgroup.

(m) any of the ACC derivatives described above with R₂ hydroxyl, —NH₂,or methoxy, except R₂ is not —OH, substituted alkoxy, alkenyloxy,alkinyloxy, cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H oran acyl group and R₇ is methyl or R₁ is methyl and R₇ is H or an acylgroup.

(n) any of the ACC derivatives described above with R₇ being —H and R₁being —H or lower alkyl.

(o) any of the ACC derivatives described above with R₇ being —H and R₁being —H or methyl.

(p) R₇ is —H; R₁ is —H, C₁-C₄ alkyl; and R₂ is —OH, —OR³ or —NR⁴R⁵,except R₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy,cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H or an acylgroup and R₇ is methyl or R₁ is methyl and R₇ is H or an acyl group;where R³ is C₁-C₄ alkyl; and R⁴ and R⁵ are independently selected fromthe group consisting of —H and C₁-C₄ alkyl.

(q) R₇ is —H; R₁ is —H or methyl; and R₂ is —OH, —NH₂ or —OMe, except R₂is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy,—NH₂, alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ ismethyl or R₁ is methyl and R₇ is H or an acyl group.

In another method described herein of inhibiting ethylene production ina plant or plant part, the plant or plant part is contacted with aneffective amount of an ACC derivative selected from

The ACC derivative may be ACC-NH₂ or N-Me-ACC.

As used herein, an alkyl group can be a straight chained or branchedalkyl group, and may include one or more cyclic alkyl moieties. As usedherein, an aryl group and a heteroaryl group may be single ring or fusedring groups. Heteroaryl groups may contain one or more heteroatoms.

In one method described herein of inhibiting ethylene production in aplant or plant part, the plant or plant part is contacted with aneffective amount of an acyclic compound of the general formula R₈—C(H,NH₂)—CO₂H, where R₈ may include any of the R₁ to R₇ groups listed above.

R₈—C(H, NH₂)—CO₂H is the general formula for an amino acid. Depending onthe R₅ group chosen, the resulting compound may be one of the standard20 amino acids found naturally; it may be a non-naturally occurringamino acid or it may be a modified amino acid. Non-naturally occurringamino acids are generally synthesized chemically. Naturally occurringamino acids may be purified from natural sources such as plants, fungi,bacteria, or yeast, or they may be synthesized chemically.

In yet another method described herein of inhibiting ethylene productionin a plant or plant part, the plant or plant part is contacted with aneffective amount of an acyclic compound of the formula R₈—C(H, NH₂)—CO₂Hselected from α-aminoisobutyric acid (AIB), D-alanine, and glycine.

In one method described herein, the plant or plant part in whichethylene production is inhibited may be but is not limited to a fruit, avegetable, a leaf, a branch, a flower, a root or a stem.

In one method described herein, the ACC derivative or acyclic compoundis in a solution. In one method the solution is an aqueous solution. Inone method described herein, the solution is sprayed on the plant orplant part. In another method described herein, the plant or plant partis immersed or partially immersed in the solution. In another methoddescribed herein, the solution is converted to a fog and then applied.In another method described herein, the solution is vaporized and thenapplied.

In one method described herein, ethylene production is inhibited inapples, pears, stone fruits, avocado, citrus, berries, tomatoes, cherrytomatoes, bananas, cucurbits or grapes. Other plant or plant parts inwhich ethylene production may be inhibited include but are not limitedto hard/soft fruits, vegetables, antisprouting, yellowing of greenbeans, lettuce, spinach, leafy vegetables, and broccoli. In one methoddescribed herein, the plant or plant part is treated before harvesting.In another method described herein, the plant or plant part is treatedafter harvesting.

Also described herein is a method of delaying the opening of a cutflower. In one such method a cut flower is contacted with a compoundhaving the following structure:

where R₁, R₂, and R₇ are as described above.

In one method of delaying the opening of a cut flower, the ACCderivative is selected from

In another such method, a cut flower is contacted with an acycliccompound of the general formula R₈—C(H, NH₂)—CO₂H, where R₈ is asdescribed above.

In one method of delaying the opening of a cut flower, an acycliccompound selected from α-aminoisobutyric acid (AIB), D-alanine, andglycine is used.

In one method of delaying the opening of a cut flower the stem end ofthe cut flower is immersed in an aqueous solution of the ACC derivativeor acyclic compound.

Also described herein is a kit for preserving a flower. The kit containsan ACC derivative having the structure:

where R₁, R₂, and R₇ are as described above.

In one kit, the ACC derivative is selected from

Another kit can contain an acyclic compound of the general formulaR₈—C(H, NH₂)—CO₂H, where R₈ is as described above.

In one kit, an acyclic compound selected from α-aminoisobutyric acid(AIB), D-alanine, and glycine is used.

The kit may be useful both preharvest and postharvest. In one kitdescribed herein, the kit further includes a floral preservative. In onekit described herein, the floral preservative includes one or more of abiocide, an acid, and a sugar. Acids that may be used include but arenot limited to acetic acid, hydrochloric acid, sulfuric acid, citricacid or acetylsalicylic acid.

Biocides that may be used include but are not limited to sodiumhypochlorite. Sugars that may be used include but are not limited todextrose. In one kit described herein, the ACC derivative or acycliccompound and the floral preservative are a solid formulation or anaqueous solution.

The kit may be useful in combination treatments with other ethyleneinhibitors.

Also described herein is a method of delaying the ripening of a fruit orvegetable by contacting the fruit or vegetable with an effective amountof an ACC derivative having the formula:

where R₁, R₂, and R₇ are as described above.

In another method of delaying the ripening of a fruit or vegetable, theACC derivative is selected from

Another method of delaying the ripening of a fruit or vegetable utilizesan acyclic compound of the general formula R₈—C(H, NH₂)—CO₂H, where R₈is as described above.

In another method of delaying the ripening of a fruit or vegetable, anacyclic compound selected from α-aminoisobutyric acid (AIB), D-alanine,and glycine is used.

In one method described herein, the fruit or vegetable for whichripening is delayed is selected from apples, pears, stone fruits,avocado, citrus, berries, tomatoes, cherry tomatoes, bananas, cucurbitsor grapes. In one method, the fruit or vegetable is treated preharvest.In one method, the fruit or vegetable is treated post harvest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the postulated initiation of the reaction catalyzed byACCO. As indicated, an activated iron-oxo species abstracts an electronfrom the amine of a substrate to form a radical cation. This is followedby a structural rearrangement of ACC that ultimately leads to ethylene.

FIG. 2 shows the full postulated mechanism for ACCO. First, substratebinds to the iron, which initiates O₂ bending and activation.Subsequently, substrate is oxidized by one electron leading to breakdownof the initial structure. Alternatively, ascorbate (ASC) can enter andreduce the activated iron center. This produces the uncoupled reaction,i.e., O₂ uptake without ethylene production.

FIG. 3 shows that, for some substrates, the initial one electronoxidation of the amine can lead to decarboxylation.

DETAILED DESCRIPTION

1-aminocyclopropane-1-carboxylic acid Oxidase (ACCO)

1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a member of thenon-heme iron enzyme family which is characterized by a 2-histidine-1carboxylic acid iron-binding motif. ACCO catalyses the last step in theethylene biosynthetic pathway: the two electron oxidation of1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, carbon dioxide,and cyanide. That reaction requires the simultaneous reduction ofmolecular oxygen to two equivalents of water and the oxidation ofascorbate to dehydroascorbate.

ACCO Inhibitors

Described herein are ACCO inhibitors of the formula:

-   -   where R₁, R₂, and R₇ may be as follows

(a) R₁ and R₇ are independently selected from H, —OH, substituted orunsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substituted or unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted or unsubstitutedC₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, or substitutedor unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; andR₂ is —OH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl, —OR³, —NR⁴R⁵, or —SR⁶,except R₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy,cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H or an acylgroup and R₇ is methyl or R₁ is methyl and R₇ is H or an acyl group;where R³ is —H, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R⁴, R⁵ and R⁶ areindependently selected from the group consisting of —H, substituted orunsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substituted or unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted or unsubstitutedC₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, or substitutedor unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl.

(b) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and R₂ is—OH, —NH₂, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; —OR³, —NR⁴R⁵, or —SR⁶, exceptR₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy,—NH₂, alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ ismethyl or R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; and R⁴, R⁵ and R⁶ areindependently selected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; or C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy.

(c) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; and R₂ is —OH, —NH₂, unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, and methoxy; C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; C₆-C₁₀ aryl substituted with a group selectedfrom halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenylsubstituted with a group selected from halo, hydroxyl, and methoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,and methoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenylsubstituted with a group selected from halo, hydroxyl, and methoxy; andR⁴, R⁵ and R⁶ are independently selected from —H, unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy.

(d) R₁ and R₇ are independently selected from H; unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆ cycloalkyl or C₃-C₆cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; and unsubstitutedC₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R₂ is —OH, —NH₂,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; or unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; and R⁴, R⁵ and R⁶ are independently selected from—H, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; and unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl.

(e) R₇ is —H; R₁ is selected from H, substituted or unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl,substituted or unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl,substituted or unsubstituted C₄-C₉ heteroaryl, or substituted orunsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R₂is —OH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl,substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl,substituted or unsubstituted C₆-C₁₀ aryl, substituted or unsubstitutedC₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted or unsubstituted C₄-C₉heteroaryl, or substituted or unsubstituted C₄-C₉ heterocycloalkyl orC₄-C₉ heterocycloalkenyl, —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,substituted or unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl, substitutedor unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, substituted orunsubstituted C₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkylor C₆-C₁₄ aralkenyl, substituted or unsubstituted C₄-C₉ heteroaryl, orsubstituted or unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; and R⁴, R⁵ and R⁶ are independently selected fromthe group consisting of —H, substituted or unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl, substituted or unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl.

(f) R₇ is —H; R₁ is selected from H; unsubstituted C₂-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; and R₂ is—OH, —NH₂, unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstitutedC₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; —OR³, —NR⁴R⁵, or —SR⁶, exceptR₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy,—NH₂, alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ ismethyl or R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a group selectedfrom halo, hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₀ arylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl substituted with agroup selected from halo, hydroxyl, amino, cyano, and lower alkoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,amino, cyano, and lower alkoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; and R⁴, R⁵ and R⁶ areindependently selected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenylsubstituted with a group selected from halo, hydroxyl, amino, cyano, andlower alkoxy; C₆-C₁₀ aryl substituted with a group selected from halo,hydroxyl, amino, cyano, and lower alkoxy; C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl substituted with a group selected from halo, hydroxyl, amino,cyano, and lower alkoxy; C₄-C₉ heteroaryl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy; or C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl substituted with a groupselected from halo, hydroxyl, amino, cyano, and lower alkoxy.

(g) R₇ is —H; R₁ is selected from H; unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; and R₂ is —OH, —NH₂, unsubstituted C₁-C₆ alkyl orC₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈cycloalkyl or C₃-C₈ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆ alkenyl substituted with agroup selected from halo, hydroxyl, and methoxy; C₃-C₈ cycloalkyl orC₃-C₈ cycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy; C₆-C₁₀ aryl substituted with a group selectedfrom halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenylsubstituted with a group selected from halo, hydroxyl, and methoxy;C₄-C₉ heteroaryl substituted with a group selected from halo, hydroxyl,and methoxy; or C₄-C₉ heterocycloalkyl or C₄-C₉ heterocycloalkenylsubstituted with a group selected from halo, hydroxyl, and methoxy; andR⁴, R⁵ and R⁶ are independently selected from —H, unsubstituted C₁-C₆alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₈ cycloalkyl or C₃-C₈cycloalkenyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; C₁-C₆ alkyl or C₂-C₆alkenyl substituted with a group selected from halo, hydroxyl, andmethoxy; C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl substituted with a groupselected from halo, hydroxyl, and methoxy; C₆-C₁₀ aryl substituted witha group selected from halo, hydroxyl, and methoxy; C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl substituted with a group selected from halo, hydroxyl,and methoxy; C₄-C₉ heteroaryl substituted with a group selected fromhalo, hydroxyl, and methoxy; and C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl substituted with a group selected from halo,hydroxyl, and methoxy.

(h) R₇ is —H; R₁ is selected from H; unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; and unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R₂ is —OH, —NH₂,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl or C₃-C₆ cycloalkenyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄ aralkenyl; unsubstituted C₄-C₉heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl or C₄-C₉heterocycloalkenyl; —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH,substituted alkoxy, alkenyloxy, alkinyloxy, cycloalkoxy, —NH₂,alkylamino, dialkylamino when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group; where R³ is —H,unsubstituted C₁-C₆ alkyl or C₂-C₆ alkenyl; unsubstituted C₃-C₆cycloalkyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl orC₆-C₁₄ aralkenyl; unsubstituted C₄-C₉ heteroaryl; or unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl; and R⁴, R⁵ and R⁶ areindependently selected from —H, unsubstituted C₁-C₆ alkyl or C₂-C₆alkenyl; unsubstituted C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl or C₆-C₁₄aralkenyl; unsubstituted C₄-C₉ heteroaryl; and unsubstituted C₄-C₉heterocycloalkyl or C₄-C₉ heterocycloalkenyl.

(i) any of the ACC derivatives described above with the heteroarylselected from pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazoyl,pyridyl, pyrazinyl, pyrimidineyl, furyl, and thienyl.

(j) any of the ACC derivatives described above with the heteroarylselected from pyrrolyl, pyridyl, and thienyl.

(k) any of the ACC derivatives described above with the heteroarylpyrrolyl.

(l) any of the ACC derivatives described above with R₂ hydroxyl, amino,or lower alkoxy, except when R₁ is H or an acyl group and R₇ is methylor R₁ is methyl and R₇ is H or an acyl group.

(m) any of the ACC derivatives described above with R₂ hydroxyl, —NH₂,or methoxy, except when R₁ is H or an acyl group and R₇ is methyl or R₁is methyl and R₇ is H or an acyl group.

(n) any of the ACC derivatives described above with R₇ being —H and R₁being —H or lower alkyl.

(o) any of the ACC derivatives described above with R₇ being —H and R₁being —H or methyl.

(p) R₇ is —H; R₁ is —H, C₁-C₄ alkyl; and R₂ is —OH, —OR³ or —NR⁴R⁵,except R₂ is not —OH, substituted alkoxy, alkenyloxy, alkinyloxy,cycloalkoxy, —NH₂, alkylamino, dialkylamino when R₁ is H or an acylgroup and R₇ is methyl or R₁ is methyl and R₇ is H or an acyl group;where R³ is C₁-C₄ alkyl; and R⁴ and R⁵ are independently selected fromthe group consisting of —H and C₁-C₄ alkyl.

(q) R₇ is —H; R₁ is —H or methyl; and R₂ is —OH, —NH₂ or —OMe, exceptwhen R₁ is H or an acyl group and R₇ is methyl or R₁ is methyl and R₇ isH or an acyl group.

Also described herein are ACCO inhibitors having the formula

Further described are ACCO inhibitors which are acyclic compounds havingthe general formula R₈—C(H, NH₂)—CO₂H, where R₈ may include any of theR₁ to R₇ groups listed above.

Further described herein are ACCO inhibitors which are acyclic compoundsselected from α-aminoisobutyric acid (AIB), D-alanine, and glycine.

ACCO inhibitors that may be used in the kits and methods describedherein can be identified by assessing a potential ACC inhibitor'sability to inhibit an ACC oxidase in vitro. Using three dimensionalstructural information of an ACC oxidase enzyme, improved inhibitors canbe designed. Such techniques for computer-aided inhibitor design areknown to one of skill in the art. Based on the known coordinating of ACCto the iron in the enzyme, substitutions can be designed that will besterically tolerated. Charged groups also may be introduced tofacilitate charge-charge interactions between the inhibitor and theenzyme. One of skill in the art also will recognize that a large numberof ACCO inhibitors can be generated using combinatorial chemistrymethods. The compounds, either pooled or individually, can be tested forthe ability to inhibit ACCO in vitro. An ACCO inhibitor can act by anumber of mechanisms.

In one mechanism, the ACCO inhibitor acts as a reversible competitive ornon-competitive inhibitor of the enzyme. In reversible competitiveinhibition, the inhibitor binds to the same binding site (the activesite) as the enzyme's natural substrate. By competing for binding, theaction of the enzyme toward its natural substrate is inhibited. Theinhibitor molecule may undergo reaction while bound to the enzyme or maynot, depending on the structure of the inhibitor molecule. In eithercase, the interaction is reversible as the inhibitor (modified or not)is released from the enzyme, allowing the enzyme to undergo catalysis orbe subject to further inhibition depending on whether the naturalsubstrate or an inhibitor molecule binds in the active site. Competitiveinhibition is characterized by the ability to overcome inhibition byraising the concentration of the natural substrate, while maintainingthe concentration of the inhibitor constant.

Another form of inhibition is non-competitive inhibition. Innon-competitive inhibition, the inhibitor binds to a site distinct fromthe site bound by the natural substrate. Thus, in this type ofinhibition, the inhibition can not be overcome by raising theconcentration of the natural substrate.

In one method, ACCO inhibitors can be acyclic compounds such asα-aminoisobutyric acid (AIB), D-alanine, and glycine. These acycliccompounds compete with ACC for binding to the ACCO active site. Uponbinding, these compounds undergo a simple decarboxylation reaction torelease compounds such as acetone, acetaldehyde, and CO₂. By binding tothe ACCO active site, these acyclic compounds are able to inhibit theability of ACCO to generate ethylene from its natural substrate, ACC.Because α-aminoisobutyric acid (AIB), D-alanine, and glycine arerelatively non-toxic to plants, these compounds can be applied to plantsand plant parts at the higher concentrations necessary to compensate forthe less effective binding of these compounds to the ACCO enzyme, asindicated by their higher Km values. (See Table 1 below.)

Many of the acyclic compounds are standard or modified amino acids whichshould be permeable to plant tissues and thus be accessible to the ACCOenzyme. Other variants of the general formula R₈—C(H, NH₂)—CO₂H willalso be useful in the practice of this invention, depending on factorssuch as cell permeability and any potential side effects.

In another mechanism, an ACCO inhibitor uncouples oxygen activation frominhibitor/substrate activation. In another mechanism, the ACCO inhibitorbinds to the enzyme and covalently modifies the enzyme, acting as asuicide inhibitor.

The invention includes the compounds described herein or incorporated byreference herein, including any and all stereoisomers, salts, hydratesand solvates of the compounds described herein or incorporated byreference herein. The invention also includes the compounds describedherein or incorporated by reference herein in their non-salt,non-hydrate/non-solvate form. Thus, while some compounds disclosedherein are depicted as salts, it is to be understood that the disclosureembraces all other salts, hydrates, and solvates of the compoundsdepicted therein, as well as the non-salt, non-hydrate/non-solvate formof the compound. Particularly preferred are biologically acceptablesalts. Biologically acceptable salts are those salts which retain thebiological activity of the free compounds and which are not biologicallyor otherwise undesirable. The desired salt of a basic compound may beprepared by methods known to those of skill in the art by treating thecompound with an acid; such a salt can be the product of a reactionproducing the compound. Examples of inorganic acids include, but are notlimited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, and phosphoric acid. Examples of organic acids include, but arenot limited to, formic acid, acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, sulfonic acids, and salicylic acid. Salts of basiccompounds with amino acids, such as aspartate salts and glutamate salts,can also be prepared. The desired salt of an acidic compound can beprepared by methods known to those of skill in the art by treating thecompound with a base; such a salt can be the product of a reactionproducing the compound. Examples of inorganic salts of acid compoundsinclude, but are not limited to, alkali metal and alkaline earth salts,such as sodium salts, potassium salts, magnesium salts, and calciumsalts; ammonium salts; and aluminum salts. Examples of organic salts ofacid compounds include, but are not limited to, procaine,dicyclohexylamine, dibenzylamine, N-ethylpiperidine,N,N′-dibenzylethylenediamine, and triethylamine salts. Salts of acidiccompounds with amino acids, such as lysine salts, can also be prepared.Examples of solvates include, but are not limited to, hydrates,hemihydrates (½H₂O), dihydrates, trihydrates, and alcoholates such asmethanolates and ethanolates.

The invention also includes all polymorphs, crystalline forms, andnon-crystalline forms of the compounds disclosed herein.

The invention also includes all stereoisomers of the compounds disclosedherein, including diastereomers and enantiomers in isolated form, aswell as mixtures of stereoisomers in any proportion, including, but notlimited to, racemic mixtures. Unless stereochemistry is explicitlyindicated in a structure, the structure is intended to embrace allpossible stereoisomers of the compound depicted.

Cut Flowers

Described herein is a method by which a cut flower can be preserved orits opening prevented or delayed by contacting the cut flower with asolution of an ACCO inhibitor as described herein. In one method, theflower and supporting stem are cut and the cut end of the stem is placedin an aqueous solution of an ACCO inhibitor. The solution can have oneor more other floral preservatives in addition to the ACCO inhibitor.When opening of the flower is desired, the flower can be placed in waterthat does not contain the ACCO inhibitor, or that contains a lowerconcentration of the ACCO inhibitor. The ACCO inhibitors also can beused to preserve a flower that has already opened. For example, alreadyopened flowers can be placed in water containing an ACCO inhibitor todelay senescence (e.g., wilting or dying) of the flower. The ACCOinhibitors useful for delaying senescence or preventing flower openingare present at concentrations sufficient to have the desired effect.Such effective concentrations can be determined by routineexperimentation either on plants, or based on the affinity of theinhibitor for ACCO. For example, where the inhibitor is also a substratefor ACCO, the inhibitor is present generally in a concentration of atleast about one-tenth of the Km value of the inhibitor/substrate for theenzyme. In one method described herein, the inhibitor is present in agreater concentration than the Km value for the substrate/inhibitor.

Because the ethylene biosynthesis pathway is conserved across variousplants, the methods described herein can generally be used with anyplant or flower. Examples of commercially relevant cut flowers include,but are not limited to: alstroemeria, carnations, chrysanthemums,delphinium, gerbera daisy, gladioli, iris, lilies, lisianthus, orchids,roses, snapdragons, and tulips.

Floral Preservatives

A number of floral preservatives are known to one of skill in the art.Floral preservatives include, for example, sugars, for example dextrose,sucrose and the like. Flower preservatives also include acids (and saltsthereof), for example, citric acid, acetylsalicylic acid, hydrochloricacid, sulfuric acid, and the like. Floral preservatives also includebiocides capable of inhibiting bacterial or fungal growth on the cutflower stem. The biocide used may be sodium hypochlorite. Floralpreservatives used may include a combination of one or more of the aboveacid, biocide and sugar.

Kits

An ACCO inhibitor can be packaged in a kit with a floral preservative.The kit can include the ACCO inhibitor and floral preservative as asolid (e.g. an amorphous or crystalline powder) suitable for making asolution having a concentration suitable for the storage of cut flowers.

A kit also can be sold as a concentrated solution which can be dilutedto give the effective solution. In one kit described herein, the floralpreservative includes at least one of a biocide, an acid and a sugar. Inanother kit described herein, the kit contains the ACCO inhibitor and atleast two of a biocide, an acid and a sugar. In another kit describedherein, the kit contains the ACCO inhibitor and a biocide, an acid and asugar.

Fruits and Vegetables

When used with an edible fruit or vegetable, the fruit or vegetable iseither submerged in or sprayed with a solution of an ACCO inhibitor. Forsuch usage, in one method described herein the inhibitor is tested forany toxicity, such that any ACCO inhibitor residue present on suchfruits or vegetable will not pose a health hazard. In one methoddescribed herein, a toxic ACCO inhibitor may be used which may be laterremoved. Suitable fruits and vegetables for treatment include, but arenot limited to, members of the apple family, the pear family, stonefruits, avocado, citrus, berries, tomatoes, cherry tomatoes, bananas,cucurbits, grapes and others.

The following non-limiting examples were carried out demonstrating themethods described herein.

Synthesis of ACCO inhibitor

The ACC derivatives described herein may be prepared by a variety ofmethods as is known in the art. One example of methods that may be usedis described in US patent application 2001/0041700, the contents ofwhich is incorporated herein by reference in its entirety. The ACCderivatives described herein may be prepared as described in theexamples below.

EXAMPLES Example 1 Materials

All reagents were purchased from Sigma or Aldrich unless otherwiseindicated. All radiochemicals were purchased from American RadiolabeledChemicals except for [1-¹⁴C]-Gly, which was purchased from ICNBiomedicals. Purity of radiolabeled compounds was determined by thinlayer chromatography. A known amount of each compound was diluted with anonradiolabeled standard and spotted on a silica plate. Solvent:n-butanol: acetic acid:water (4:1:1). The compound was localized bystaining with either iodine or ninhydrin. Spots were scrapped off into ascintillation vial and the radioactivity determined. Purity wasdetermined by measuring the amount of radioactivity that ran with theknown non-radiolabeled standard as compared to that of an undevelopedplate. All radiolabeled compounds were >98% pure except[carboxyl-¹⁴C]-ACC, which was ˜20% pure.

Overexpression and Purification of ACCO.

ACCO from Lycopersicon esculentum (ACO1) was produced in Escherichiacoli strain BL21 (DE3)pLysS and purified by a two-column purificationprocedure as previously described (Thrower, J. S. et al., 2001).

Synthesis of N-BOC-ACC

N-BOC-ACC was synthesized by (1) adding ACC to water dioxane (1:1)mixture, (2) adding TEA to the mixture, (3) stirring until allcomponents dissolved, (4) adding BOC-ON to the mixture, and (5) stirringthe mixture overnight.

Synthesis of N-Me-ACC and N-[¹⁴C-methyl]-ACC

MeACC was synthesized as follows. Anal. Calculated for C₅H₉NO₂:115.06318. Found: 115.06333. N-[¹⁴C-methyl]-ACC was synthesized by thesame procedure except using [¹⁴C]—CH₃I diluted with nonradiolabeled CH₃Ito a specific activity of 10 μCi/mmol. Purity of N-[¹⁴C-methyl]-ACC wasdetermined by TLC as described above, and was found to be >98% pure.

Synthesis of ACCNH₂

N-Boc-ACC was prepared as described above. N-Boc-ACC (0.2 g, 1 mmol) andHOBT (1-hydroxybenzotriazole hydrate) (0.135 g, 1 mmol) were added toanhydrous DMF (dimethylformamide) (5 mL) and stirred on ice untildissolved. EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride) (0.192 g, 1 mmol) was added to the DMF solution andstirred on ice for 3 h. Anhydrous 7 N NH₃ in methanol (1.5 mL, ˜10 mmol)and TEA (triethylamine) (0.31 mL, ˜2 mmol) were added to the coolmixture and stirred on ice for 1 h followed by overnight at ambienttemperature. The DMF was evaporated and the resulting oil wasresuspended in 20 mL EtOAc and washed with saturated NaHO3, 0.5 M citricacid, and brine. The organic layer was dried and evaporated to yield ayellow residue. TFA (trifluoroacetic acid) (0.5 mL) was added to theresidue, stirred at ambient temperature for 2 h, and evaporated. 1 M HCl(5 mL) was added, stirred briefly, and evaporated to yield a yellow oil.The oil was resuspended in 10 mL ether. The precipitated ACCNH₂ wasfiltered and dried to yield 134 mg (30%) of white solid.

Initial Velocity Assays

Initial velocities were measured by the rate of oxygen consumption at25° C., pH 7.2, using a YSI model 5300 biological oxygen monitor aspreviously described (Thrower 2001) with minor modifications. Standardreaction mixture (1 ml) contained 100 mM MOPS, pH 7.2, 20 mM NaHCO3, 100mM NaCl, 20 mM sodium ascorbate, 0.1 mg/ml BSA, and ACC or substrateanalog at various concentrations. Oxygen concentrations were kept atatmospheric concentration and determined using the oxygen monitor thatwas calibrated with air saturated water (258 μM oxygen at 25° C.).Reactions were initiated with 2 μL ACCO reconstituted with equimolarFe(NH₄)₂(SO₄)₂. The concentration of ACCO is as indicated in figurelegends. Data from initial velocity experiments with varying substrateconcentrations were fitted to the Michaelis-Menton equation using theprogram Kalideograph.

TABLE 1 Kinetic Parameters for ACC Oxidase with Various SubstrateAnalogs^(a) Substrate V_(max) (min⁻¹) K_(m) (mm) Substrate V_(max)(min⁻¹) K_(m) (mm) ACC 36.4 ± 1.4 0.099 ± 0.018 N-Me-ACC 12.2 ± 0.50.107 ± 0.021 AIB 22.1 ± 1.6 0.92 ± 0.29 ACC-NH₂ 11.3 ± 0.6 0.214 ±0.047 D-Alanine 29.8 ± 0.9 4.42 ± 0.49 ACC-OMe 27.3 ± 1.7  2.76 ± 0.55Glycine  9.5 ± 0.6 1.0 ± 0.4 ^(a)Kinetic parameters were determined bymeasuring initial velocities at various concentrations of substratewhile maintaining ascorbate and oxygen at a constant concentration.[Ascorbate] = 30 mM, [O₂] = 258 μM, and [CO₂/bicarbonate] = 20 mM.

As can be seen from the above data, the K_(m) values for MeACC andACCNH₂ were not significantly altered compared to ACC, while the K_(m)for ACC-OMe was increased ˜28-fold. Spectroscopic studies haveidentified that both the amine and carboxylate groups of ACC are ligandsto the active site Fe(II) (Rocklin et al., 1999). It is possible thatboth MeACC and ACCNH₂ would still form a bidentate ligand to the Fe(II)center, and thus result in an equal or greater affinity for the enzyme.Likewise, ACC-OMe may be capable of forming a bidentate ligand to theFe(II) center, but one possibility for the observed decreased affinityis that it does not form a bidentate ligand. In one method foridentifying ACCO inhibitors for us in the methods described herein, theACCO inhibitor will be able to form such a bidentate interaction withthe Fe(II) center.

As can also be determined from Table 1, the K_(m) of the acycliccompounds, α-aminoisobutyric acid (AIB), D-alanine, and glycine, areincreased by factors of 9.3, 44.7, and 10.1, respectively, when comparedwith the natural substrate, ACC. The V_(max) values for the acycliccompounds show a less dramatic difference than that observed with ACC asthe substrate.

The dependence of the initial velocity of oxygen consumption byACCO:Fe(II) on the concentration of ACC or several substrate analogs wasdetermined (Table 1). Either cyclic substrate analogs (includinginhibitor Me-ACC, ACC-NH₂, or ACC-OMe) or acyclic substrate analogs(AIB, D-Ala, or Gly) were used. The presence of any one of the substrateanalogs resulted in the immediate increase of O₂ consumption byACCO:Fe(II) and, as expected, the dependence of the initial rate onsubstrate concentration followed Michaelis-Menton kinetics. Ascorbateand oxygen concentrations were kept constant at 30 mM and ˜258 μM,respectively. These concentrations were previously shown to besaturating when ACC was the substrate (Thrower et al., 2001), and wereassumed to be at saturating concentrations when substrates other thanACC were used. Additionally, measurement of the K_(m) values for O₂ orascorbate in the presence of saturating concentrations of MeACC resultedin similar K_(m) values compared to those when ACC was the substrate.This result indicated that slight perturbations in the substratestructure did not affect the Km values of O₂ or ascorbate for theenzyme.

Ethylene and CO₂ Trapping Assays

For detection of CO₂, trace radiolabeled compound was used asappropriate. A small vial containing the reaction mixture minus enzymewas placed along side of another small vial containing the ethylene orCO₂ trap. Both vials were placed in a large vial, which was capped witha septum. ACCO:Fe(II) was introduced into the reaction vial with asyringe and both the reaction vial and trap vial were stirred for 1 h.For [¹⁴C]—CO₂ trapping, a 1 mL solution of freshly prepared 10 M NaOHwas used as the trapping agent. At the end of the reaction, the trap wasremoved and added to 15 mL scintillation fluid (Hionic-Fluor, PerkinElmer) and radioactivity determined by scintillation counting. Forethylene, a 500 mL solution of Br₂ in CCl₄ was used as the trap. A smallamount of 2,5-dibromohexane was added to the trap as an internalstandard. At the completion of the reaction, a small excess of2,3-dimethyl-2-butene was added to the trap to quench unreacted Br₂, andthe trap was analyzed by GC-MS. Dibromoethane purchased from Aldrich wasused as a standard.

Stoichiometry of O₂ Consumption to CO₂ Production

Oxygen consumption was measured using a YSI model 5300 biological oxygenmonitor as described above. Reactions contained D-[1-¹⁴C]-Ala,[carboxyl-¹⁴C]-ACC, α-[1-¹⁴C]-AIB, or [1-¹⁴C]-Gly in trace amounts.Substrates were kept at saturating concentrations (at least 10-foldabove Km), 30 mM ascorbate, and ambient O₂ concentrations. Reactions (1mL) were initiated with a pre-incubated mixture of ACCO:Fe(II) in a 2:1ratio. This was done to minimize free Fe(II) in solution. Finalconcentrations were typically 10 μM ACCO and 5 μM Fe(II). Reactions werequenched by addition of 3 μL of 0.1 M3-[2-Pyridyl]-5,6-diphenyl-1,2,4-triazine-4,4′-disulfonic acid(FERROZINE®) and the O₂ consumption rate was allowed to stabilize. Thereaction (0.8 mL) was removed with a gas-tight syringe and injected intoa small vial containing 0.1 mL 10% HCl that was inside a larger vialcontaining the CO₂ trap (see above). The acidified reaction mixture andthe CO₂ trap were stirred for 2 h, and radioactivity from [¹⁴C]—CO₂ wasdetermined as described above. Controls measuring the efficiency of theCO₂ trap were done by injecting a known amount of [¹⁴C]—NaHCO₃ into thevial containing acid, as above, and measuring the amount of [¹⁴C]—CO₂recovered. Efficiency of trapping was routinely ˜98%.

O₂ consumption was measured using an Ocean Optics FOXY fiber opticoxygen sensor, with the fiber optic probe inserted through a syringeinto an airtight septum, thus preventing ethylene escape. Substrateswere kept at saturating concentrations (at least 10-fold above K_(m)),30 mM ascorbate, and ambient O₂ concentrations. ACCO:Fe(II) (finalconcentrations 10 μM ACCO and 5 μM Fe(II)) was injected by syringe intoa ½ dram vial covered with a septum with no headspace. The reaction wasquenched as described above. A 10 mL syringe filled with air was used totransfer the reaction mixture to a 10 mL test tube covered with a septumusing a cannula. The test tube was stirred vigorously for at least 4hours before ethylene analysis. Ethylene was measured by injecting 2 mLof the headspace onto a GC equipped with a RT-Alumina GC column (finalsample size was 250 μL). Ethylene was separated using ultrapure helium(grade 5) as the carrier gas at 40° C. detected by flame ionization(FID). The data were collected and the peak area was determined.Calibration for ethylene was done with Scotty IV mix 3 hydrocarbonstandard (Scotty Specialty Gases). The ppm of ethylene was converted tonanomoles of ethylene per unit volume using the ideal gas law. Dilutionof ethylene during sampling was taken into account in the calculations.

The results show that ACCO consumes O₂ in the presence of either AIB orD-Ala at a rate comparable to that with ACC (Table 1). However, this isnot indicative of substrate turnover. There have been several examplesof related non-heme iron enzymes that activate O₂ without correspondingsubstrate turnover in what is called an “uncoupled” reaction (Holme etal., 1982, and Liu et al., 2001). In some cases, significant uncouplingeven occurs during normal catalysis with the physiological substrate(Holme et al., 1982) and leads to either reversible or irreversibleenzyme inactivation (Barlow et al., 1997, and Liu et al., 2001).

The ability of ACCO to turnover ACC or the cyclic substrate analogs(MeACC, ACC-NH₂, or ACC-OMe) was determined by the formation ofethylene. Each substrate analog or ACC was incubated for 1 hr withACCO:Fe(II) in a closed reaction vial containing an ethylene trap thatwould convert ethylene to dibromoethane. At the end of the reactiontime, the ethylene trap was quenched and analyzed for dibromoethane byGC-MS. Reactions with BSA/Fe(II) were used as a control fornon-enzymatic ethylene formation. The formation of ethylene was detectedin the reaction vials of ACC and all the cyclic analogs in the presenceof ACCO:Fe(II). No ethylene was detected in any of the BSA/Fe(II)reactions. The amount of ethylene formed during the reaction time fromthe cyclic analogs was lower than that from the reaction with ACC(MeACC=14%, ACCNH₂=13%, ACC-OMe=79%). If the rates of O₂ consumption forthe reactions with the different substrates are considered, the k_(cat)for ACC-OMe is approximately 75% of that for ACC, which would beconsistent with the amount of ethylene produced during the time courseof the reaction. However, ACCNH₂ and MeACC had k_(cat) values ˜30%slower than the k_(cat) for ACC, but only produced about half thepredicted ethylene during the reaction time course. Although the purityof both MeACC and ACCNH₂ by elemental analysis was determined >95%, itis possible that a small amount of contaminating ACC could beresponsible for the ethylene formed in these reactions. To determinewhether ethylene was being formed from the substrate analog and not fromcontaminating ACC, reactions with each analog were done in the presenceof increasing concentration of ACCO:Fe(II). In the case of a smallamount of contamination where the ACC would be quickly depleted duringthe reaction, the result would be a non-linear dependence of ethyleneproduction with increasing ACCO concentration. For ACC and all cyclicanalogs, the amount of ethylene production with increasing concentrationof ACCO showed a linear dependence, indicating that ethylene productionwas due to substrate analog turnover rather than ACC contamination.

Example 2 Inhibition of ACCO by N-Me-ACC

Using the following protocol, it was demonstrated that radiolabeledN-methyl-ACC irreversibly labels ACCO in a short period of time.Radiolabeled N-Me-ACC was incubated with ACCO (approximately equalconcentrations) in standard reaction conditions. The reaction wasquenched using Ferrozine. The solution was placed in a Millipore 10KNMWL Ultrafree®-MC Centrifugal Filter Unit and centrifuged for 15 min.at 14K rpm. The remaining protein (˜20 μL) was washed with 3×1 mL ofeither water, reaction buffer, or 0.1% TFA by repeated centrifugation.The protein was collected and radioactivity determined by scintillationcounting in 12 mL EcoLite⁺ scintillation fluid.

Stoichiometry of O₂ Consumed to Product Formed

The extent of uncoupling for ACCO with both cyclic and acyclic substrateanalogs was determined by comparing the amount of O₂ consumed andproduct, either ethylene or CO₂, formed. All reactions were carried outat saturating substrate and ascorbate concentrations to minimizepotential O₂ reaction in the absence of substrate. Enzyme wasreconstituted with 0.6 equivalents of Fe(II) and kept at highconcentration (12-fold higher than Kd) to minimize free Fe(II) in thereaction. O₂ measurements were quenched during or just as the linearphase of the reaction began to fall off due to either loss of Fe(II)from the enzyme or possible enzyme inactivation. The results distinguishbetween amounts of uncoupling.

For cyclic substrate analogs and ACC, the stoichiometry was determinedby comparing O₂ consumption with ethylene formation by GC-FID. For theacyclic substrate analogs that were shown to release CO₂ as a product,as well as ACC, O₂ consumption was compared with the formation of[¹⁴C]—CO₂. Ratios of O₂ consumption to product formation are shown inTable 3. The stoichiometry of O₂ consumed to ethylene produced for ACCwas 1.44 and to CO₂ produced was 1.18, which is identical to thatreported by Dong, et. al (Dong et al., 1992).

All cyclic and acyclic substrates showed some increased level ofuncoupling over ACC. However, with the cyclic substrates there was nocorrelation of the extent of uncoupling and whether the amine (MeACC) orthe carboxylate (ACCNH₂ or ACC-OMe) had been perturbed. In the case ofthe acyclic substrates, a more definite pattern emerged where the extentof uncoupling was affected by the substrate structure. Breaking thecyclic ring but maintaining both methyl groups in AIB resulted in someincreased uncoupling over the ACC reaction (1.7 for AIB versus 1.2 forACC). However, each successive removal of a methyl group from AIB to Alaand then to Gly resulted in a further increase in the extent ofuncoupling, with Gly having the most extreme affect of only one of aboutevery 21 reactions with O₂ resulting in Gly turnover.

TABLE 2 Stoichiometry of Oxygen Reduction Catalyzed by ACC Oxidase withVarious Substrates Ratio O₂ consumed to product formed Substrate O₂:CO₂O₂:ethylene Products detected ACC 1.18 ± 0.17 1.44 ± 0.22 Ethylene, CO₂N-Me-ACC — 3.17 ± 0.86 Ethylene ACC-NH₂ — 4.40 ± 1.56 Ethylene ACC-OMe —1.81 ± 0.72 Ethylene AIB 1.68 ± 0.12 — Acetone, CO₂ D-Alanine 3.46 ±0.51 — Acetaldehyde, CO₂ Glycine 21.4 ± 7.9  — CO₂

Examples 3-5 are prophetic examples.

Example 3 Closed Flower Storage

A dozen lilies are cut at similar stages of floral development. The cutend of each of 6 lilies is submerged in 100 mL of 10 mM N-methyl-ACC orACC-NH₂. Alternatively, the cut ends can be submerged into 100 mL of asolution containing an appropriate concentration of α-aminoisobutyricacid (AIB), D-alanine, and glycine. Other concentrations of theseinhibitor compounds may be suitable for a given plant variety, and canbe determined by trial and error. The cut ends of the remaining sixlilies are submerged in untreated water. The 12 lilies are placed in acold room at 5° C. in the dark. After two weeks in the cold room, theflowers are transferred to room temperature, and the time until floralopening is measured.

Example 4 Opened Flower Storage

A dozen lilies that were previously cut and have opened within the past24 hours are either placed in a solution of 10 mM N-methyl-ACC, 10 mMACC-NH₂, or solutions containing appropriate concentrations ofα-aminoisobutyric acid (AIB), D-alanine, glycine or plain water. Otherconcentrations of both inhibitor compounds may be suitable for a givenplant variety, and can be determined by trial and error. The flowers areallowed to stand at room temperature until the flowers have noticeablywilted. The storage solutions and water are refreshed by adding freshsolution as necessary, but at most once per day.

Example 5 Fruits and Vegetables

Harvested fruits and vegetables are sprayed with a suitableconcentration of N-methyl-ACC, ACC-NH₂, α-aminoisobutyric acid (AIB),D-alanine, glycine or water as a control. For example, 10 mM solutionsof each type of cyclic inhibitor or appropriate concentrations ofacyclic inhibitor can be applied. Fruits and vegetables includingmembers of the apple family, stone fruits, avocado, citrus, berries,tomatoes, cherry tomatoes, bananas, cucurbits, grapes and other typescan be treated as above. The solution of ACCO inhibitor is applied byspraying, drenching, or fogging and allowed to contact the subjectproduce item for a suitable amount of time before washing off. Theproper concentration of ACCO inhibitor for each type of produce can bedetermined by trial and error. The effectiveness of inhibition on agiven piece of fruit is monitored by applying the ACCO inhibitor andthen testing the subject fruit item for release of ethylene gas. Theinhibitor compound is tested for toxicity prior to being applied toproduce items that will be used for food.

REFERENCES

The following references are hereby incorporated by reference in theirentirety.

-   Barlow, J. N., Zhang, Z., John, P., Baldwin, J. E.,    Schofield, C. J. (1997) Biochemistry 36, 3563-3569.-   Bregoli, et al. (2002) Physiologia Plantarum, 114(3), 472-481.-   Brunhuber, N. M., Mort, J. L., Christoffersen, R. E.,    Reich, N. O. (2000) Biochemistry 39, 10730-10738.-   Dong, J. G., Fernández-Maculet, J. C., Yang, S. F. (1992) Proc.    Natl. Acad. Sci. USA 89, 9789-9793.-   Fernández-Maculet, J. C., Dong, J. G., Yang, S. F. (1993) Biochem.    Biophys. Res. Commun. 193, 1168-1173.-   Gibson, E, J., Zhang, Z., Baldwin, J. E., Schofield, C. J. (1998)    Phytochem. 48, 619-624.-   Griller, D., Ingold, K. (1980) Acc. Chem. Res. 13, 317-323.-   Holme, E., Lindstedt, s., Nordin, I. (1982) Biochem. Biophys. Res    Commun. 107, 518-524.-   Liu, Y., Su, L.-Y., Yang, S. F. (1984) Planta 161, 439-443.-   Liu, A., Ho, R. Y. N., Que, L. Jr. (2001) J. Am. Chem. Soc. 123,    5126-5127.-   Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M. Jr.,    Krebs, C. (2003) Biochemistry 42, 7497-7508.-   Rocklin, A. M., Tierney, D. L., Kofman, V., Brunhuber, N. M. W.,    Hoffman, B. M., Christoffersen R. E., Reich, N. O., Lipscomb, J. D.,    Que, L. Jr. (1999) Proc. Natl. Acad. Sci. USA 96, 7905-7909.-   Rocklin, A. M., Kato, K., Liu, H.-W., Que, L. Jr.,    Lipscomb, J. D. (2004) J. Biol. Inorg. Chem. 9, 171-182.-   Solomon, E. I., Burnold, T. C., Davis, M. I., Kemsley, J. N., Lee,    S.-K., Lehnert, N., Neese, R., Skulan, A. J., Yang, Y.-S.,    Zhou, J. (2000) Chem. Rev. 100, 235-349.-   Su, Z., Falvey, D. E., Yoon, U. C., Mariano, P. S. (1997) J Am.    Chem. Soc. 119, 5261-5262.-   Thrower, J. S., Blalock, R. 3^(rd), Klimnan, J. P. (2001)    Biochemistry 40, 9717-9724.-   Vaidyanathan, G., Wilson, J. W. (1989) J. Org. Chem. 54, 1815-1820.-   Zhang, W., Yeh, S.-R., Hong, S., Freccero, M., Albini, A.,    Falvey, D. E., Mariano, P. S. (1994) J. Am. Chem. Soc. 116,    4211-4220.-   Zhang, Z., Schofield, C. J., Baldwin, J. E., Thomas, P.,    John, P. (1995) Biochem. J. 307, 77-85.

All references, patents, and patent application described herein areincorporated herein in their entirety to the same extent as though eachreference, patent and patent application had been individuallyincorporated by reference.

1. A method of inhibiting ethylene production in a plant or plant part,the method comprising: contacting the plant or plant part with aneffective amount of an ACC derivative having the formula:

wherein R₁ and R₇ are independently selected from H, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₈cycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted orunsubstituted C₆-C₁₄ aralkyl, substituted or unsubstituted C₄-C₉heteroaryl, or substituted or unsubstituted C₄-C₉ heterocycloalkyl; R₂is —OH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted C₆-C₁₀aryl, substituted or unsubstituted C₆-C₁₄ aralkyl, substituted orunsubstituted C₄-C₉ heteroaryl, or substituted or unsubstituted C₄-C₉heterocycloalkyl, —OR³, —NR⁴R⁵, or —SR⁶, except R₂ is not —OH when R₁ isH and R₇ is H; R³ is —H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted C₃-C₈ cycloalkyl, substituted orunsubstituted C₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄ aralkyl,substituted or unsubstituted C₄-C₉ heteroaryl, or substituted orunsubstituted C₄-C₉ heterocycloalkyl; R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of —H, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted C₃-C₈ cycloalkyl, substitutedor unsubstituted C₆-C₁₀ aryl, substituted or unsubstituted C₆-C₁₄aralkyl, substituted or unsubstituted C₄-C₉ heteroaryl, or substitutedor unsubstituted C₄-C₉ heterocycloalkyl; and wherein said of R₁, R₂ orR₇ is a heteroaryl selected from the group consisting of pyrrolyl,imidazolyl, pyrazolyl, isothiazolyl, isoxazoyl, pyridyl, pyrazinyl,pyrimidineyl, furyl, and thienyl.
 2. The method of claim 1, wherein thesubstituted C₁-C₆ alkyl, substituted C₃-C₈ cycloalkyl, substitutedC₆-C₁₀ aryl, substituted C₆-C₁₄ aralkyl, substituted C₄-C₉ heteroaryl,and substituted C₄-C₉ heterocycloalkyl, are independently substitutedwith one or more groups selected from halo, hydroxyl, amino, cyano, andlower alkoxy.
 3. The method of claim 2, wherein the substituted C₁-C₆alkyl, substituted C₃-C₈ cycloalkyl, substituted C₆-C₁₀ aryl,substituted C₆-C₁₄ aralkyl, substituted C₄-C₉ heteroaryl, andsubstituted C₄-C₉ heterocycloalkyl, are independently substituted withone or more groups selected from halo, hydroxyl, and methoxy.
 4. Themethod of claim 1, wherein R₁ and R₇ are independently selected from H;unsubstituted C₁-C₆ alkyl; unsubstituted C₃-C₆ cycloalkyl; unsubstitutedC₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl; unsubstituted C₄-C₉heteroaryl; and unsubstituted C₄-C₉ heterocycloalkyl; and R₂ is —OH,—NH₂, unsubstituted C₁-C₆ alkyl; unsubstituted C₃-C₆ cycloalkyl;unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl; unsubstitutedC₄-C₉ heteroaryl; unsubstituted C₄-C₉ heterocycloalkyl; —OR³, —NR⁴R⁵, or—SR⁶; where R³ is —H, unsubstituted C₁-C₆ alkyl; unsubstituted C₃-C₆cycloalkyl; unsubstituted C₆-C₁₀ aryl; unsubstituted C₆-C₁₄ aralkyl;unsubstituted C₄-C₉ heteroaryl; or unsubstituted C₄-C₉ heterocycloalkyl;and R⁴, R⁵ and R⁶ are independently selected from —H, unsubstitutedC₁-C₆ alkyl; unsubstituted C₃-C₆ cycloalkyl; unsubstituted C₆-C₁₀ aryl;unsubstituted C₆-C₁₄ aralkyl; unsubstituted C₄-C₉ heteroaryl; andunsubstituted C₄-C₉ heterocycloalkyl.
 5. The method according to claim1, wherein R₇ is —H.
 6. The method according to claim 1, wherein theheteroaryl is elected from the group consisting of pyrrolyl, pyridyl,and thienyl.
 7. The method according to claim 6, wherein the heteroarylis pyrrolyl.
 8. The method according to claim 1, wherein R₂ is hydroxyl,amino, or lower alkoxy.
 9. The method according to claim 8 wherein R₂ ishydroxyl, —NH₂, or methoxy.
 10. The method according to claim 1, whereinR₇ is —H and R₁ is —H or lower alkyl.
 11. The method of claim 10,wherein R₁ is —H or methyl.
 12. The method of claim 1, wherein the plantor plant part is a fruit, a vegetable, a leaf, a branch, a flower, aroot or a stem.